Co‐culture of monocytes with autologous fragment (F) spheroids originating from malignant (M) tumour or benign (B) control mucosa of head and neck squamous cell carcinoma (HNSCC) yields interleukin (IL)‐6 and monocyte chemo‐attractant protein (MCP)‐1 secretion. This study investigates the association between this cytokine co‐culture response and prognosis. Analysis of IL‐6 and MCP‐1 content of supernatants from monocytes in vitro co‐culture with autologous MF‐ or BF‐spheroids was investigated in a cohort of HNSCC patients (n = 65) diagnosed between 1998 and 2005, all of whom were treated with curative intent by primary surgery. The IL‐6 response was expressed as a fraction of the lipopolysaccharid response of the same batch of monocytes. Recurrence, survival and causes of death were then established following the second part of 2005. MCP‐1 levels did not predict prognosis. We found that increased levels of IL‐6 from autologous monocytes in co‐culture with MF‐spheroids predicted recurrence with a hazard ratio (HR) of 1.5 [confidence interval (CI): 1.01–2.60; P = 0.05] and co‐culture with BF‐spheroids and monocytes predicted recurrence (HR = 4.17; CI: 1.54–11.29; P = 0.005). The same results where obtained in addition with TNM stage of the patients. Simultaneous analysis of BF‐ and MF‐spheroid co‐culture IL‐6 responses as well as adjustment for age and TNM stage of the patients allowed prediction of total survival (HR = 3.1; CI: 1.11–8.56; P = 0.03) based on BF co‐culture levels. IL‐6 secreted upon in vitro co‐culture with monocytes and BF‐spheroids predicts recurrence and prognosis, whereas co‐culture with monocytes and MF‐spheroids predicts recurrence.

Interaction between the immune system and cancer allows for the use of biological response modifiers, e.g. OK‐432, in cancer therapy. OK‐432, penicillin‐killed Streptococcus pyogenes, is used in treating carcinomas, but also lymphangiomas. We have studied the role of monocytes (MOs) in the immune response to OK‐432 by examining IL‐6 and tumour necrosis factor (TNF)‐α secretion after in vitro MO stimulation with OK‐432, to some extent in comparison with lipoteichoic acid (LTA) and lipopolysaccharide (LPS). LTA stimulation of whole blood gave IL‐6 but not TNF‐α secretion, as previously shown with OK‐432 stimulation, whereas both cytokines were secreted following LPS stimulation. Addition of the MAPK kinase (MAPKK) MEK inhibitor U0126 inhibited IL‐6/TNF‐α secretion in a dose‐dependent manner. Flow cytometry and to some extent Western blot (Wb) analyses showed that MAPK ERK, located downstream of MEK1/2, is predominantly phosphorylated at isolation from peripheral blood. Addition of the p38 MAP kinase inhibitor SB202190 decreased MO IL‐6/TNF‐α production upon OK‐432 stimulation in a dose‐dependent manner. Addition of the MAPK JNK inhibitor SP600125 did not systematically change the MO IL‐6/TNF‐α OK‐432 response. Flow cytometry showed that when stimulating the MOs before isolation from blood, LPS yielded ERK phosphorylation and LPS/LTA p38 phosphorylation, whereas OK‐432 had no effects on phosphorylation levels. In conclusion, we have shown that OK‐432 resembles TLR2 more than TLR4 stimulation of MOs and depends on MAPKK MEK and MAPK p38, but not on JNK phosphorylation. The MEK and p38 MO OK‐432 stimulation dependence is possibly related to the differentiation of cells of the MO lineage.

OK‐432, penicillin‐killed Streptococcus pyogenes, is used in treating lymphangiomas and carcinomas. We have studied proinflammatory interleukin (IL) secretion following OK‐432 stimulation of total blood, peripheral blood mononuclear cell (PBMC) and purified monocytes in vitro. OK‐432 stimulation of purified monocytes gave IL‐1β, IL‐1RA, IL‐6, IL‐12p40 and tumour necrosis factor (TNF)‐α response. OK‐432 stimulation of cells within blood did, however, not yield TNF‐α secretion. When PBMC or monocytes were cultured in low‐attachment wells a decreased IL secretion was observed compared to adherent cells. Inhibition of Syk kinase with piceatannol, only at high, non‐specific doses, but not PI3 kinase inhibition with LY294002 or Wortmannin, decreased monocyte IL response to OK‐432. This shows that β1–3‐integrin receptor function is not necessary for monocyte OK‐432‐stimulated TNF‐α secretion. Direct blockage of the β2‐integrin (CD18) receptor by anti‐CD18 antibody was also unable to prevent the stimulating effects of OK‐432 in human monocytes. On the other hand, Syk phosphorylation is elevated upon adherence of monocytes and this is further increased by OK‐432 stimulation, as shown by Western blot. The Fc‐receptor was also ruled out as a main receptor of the OK‐432 monocyte response. In conclusion, TNF‐α secretion is only found in monocytes removed from blood. This TNF‐α secretion is not mediated through the β1–3‐integrin receptors. OK‐432 may act as a target‐seeking substance whereby only monocytes adhered, e.g. to a tumour cell, become cytotoxic in part explaining why OK‐432 is well suited as a cancer treatment drug.

Monocyte fragment (F)-spheroid-stimulated and F-spheroid IL-6 and monocyte chemotactic protein (MCP)-1 secretion are related to inflammatory state, macrophage density and the TNM stage of patients with head and neck squamous cell carcinoma (HNSCC). Fragment (F)-spheroids from HNSCC patients in vitro secrete and stimulate autologous monocytes to secrete IL-6 and MCP-1. The aim of this investigation was to study this cytokine secretion in relation to other cytokines, spheroid composition and host factors.In series I (n=14) the densities of epithelial cells, fibroblasts and macrophages were determined in sections from F-spheroids and donor tissue. In series II (n=17) the TNM stage, donor inflammatory state, macrophage density and the secretion of F-spheroid- and monocyte F-spheroid-stimulated IL-6, MCP-1 and tumor necrosis factor (TNF)-alpha were determined. Epithelial cells were partly replaced by interstitial tissue during spheroid formation. Malignant (M) F-spheroids secreted more MCP-1 than benign (B) F-spheroids. No F-spheroid secreted measurable amounts of TNF-alpha. Monocytes secreted more IL-6 when co-cultured with MF- compared to BF-spheroids. Monocyte IL-6 MF- and MCP-1 MB-spheroid-stimulated secretion correlated with macrophage density. In addition, there was an association between MF- and BF-spheroid-stimulated monocyte cytokine secretion, as well as between BF- and MF-spheroid-stimulated MCP-1 secretion. An inverse relation was also noted between the erythrocyte sedimentation rate at monocyte harvest and the monocyte MCP-1 F-spheroid responses.